Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Photolyases contain two chromophores, flavin plus either methenyltetrahydrofolate (MTHF) or 8-OH-5-deazaflavin (HDF). Amino acid sequence comparison reveals that all photolyases sequenced to date have extensive sequence homology in the carboxyl-terminal half; in the amino-terminal region the folate and deazaflavin class enzymes are more homologous to other members of the same class. This modular arrangement of sequence homologies suggests that the amino-terminal half of photolyase is involved in MTHF or HDF binding whereas the carboxyl-terminal half carries the flavin binding site. In this study we attempted to identify such structural domains of yeast photolyase by partial proteolysis and gene fusion techniques. Partial digestion with chymotrypsin yielded an amino-terminal 34-kDa fragment containing tightly bound MTHF and a carboxyl-terminal 20-kDa polypeptide which lacked chromophore or DNA binding activity. However, a fusion protein carrying the carboxyl-terminal 275 amino acids of yeast photolyase bound specifically to FAD but not to MTHF or DNA. We conclude that the amino-terminal half of yeast photolyase constitutes the folate binding domain and that the carboxyl-terminal half carries the flavin binding site.
...
PMID:Identification of chromophore binding domains of yeast DNA photolyase. 173 47

Native DNA photolyase, as isolated from Escherichia coli, contains a neutral flavin radical (FADH.) plus a pterin chromophore (5,10-methenyltetrahydropteroylpolyglutamate) and can be converted to its physiologically significant form by reduction of FADH. to fully reduced flavin (FADH2) with dithionite or by photoreduction. Either FADH2 or the pterin chromophore in dithionite-reduced native enzyme can function as a sensitizer in catalysis. Various enzyme forms (EFADox, EFADH., EFADH2, EPteFADox, EPteFADH., EPteFADH2, EPte) containing stoichiometric amounts of FAD in either of its three oxidation states and/or 5,10-methenyltetrahydrofolate (Pte) have been prepared in reconstitution experiments. Studies with EFADox and EPte showed that these preparations retained the ability to bind the missing chromophore. The results suggest that there could be considerable flexibility in the biological assembly of holoenzyme since the order of binding of the enzyme's chromophores is apparently unimportant, the binding of FAD is unaffected by its redox state, and enzyme preparations containing only one chromophore are reasonably stable. The same catalytic properties are observed with dithionite-reduced native enzyme or EFADH2. These preparations do not exhibit a lag in catalytic assays whereas lags are observed with preparations containing FADox or FADH. in the presence or absence of pterin. Photochemical studies show that these lags can be attributed to enzyme activation under assay conditions in a reaction involving photoreduction of enzyme-bound FADox or FADH. to FADH2. EPte is catalytically inactive, but catalytic activity is restored upon reconstitution of EPte with FADox. The results show that pterin is not required for dimer repair when FADH2 acts as the sensitizer but that FADH2 is required when dimer repair is initiated by excitation of the pterin chromophore. The relative intensity of pterin fluorescence in EPte, EPteFADH., EPteFADox, or EPteFADH2 has been used to estimate the efficiency of pterin singlet quenching by FADH. (93%), FADox (90%), or FADH2 (58%). Energy transfer from the excited pterin to flavin is energetically feasible and may account for the observed quenching of pterin fluorescence and also explain why photoreduction of FADox or FADH. is accelerated by the pterin chromophore. An irreversible photobleaching of the pterin chromophore is accelerated by FADH2 in a reaction that is accompanied by a transient oxidation of FADH2 to FADH.. Both pterin bleaching and FADH2 oxidation are inhibited by substrate.
...
PMID:Chromophore function and interaction in Escherichia coli DNA photolyase: reconstitution of the apoenzyme with pterin and/or flavin derivatives. 240 8

DNA photolyase from Escherichia coli contains both flavin and pterin. However, the isolated enzyme is depleted with respect to the pterin chromophore (0.5 mol of pterin/mol of flavin). The extinction coefficient of the pterin chromophore at 360 nm is underestimated by a method used in earlier studies which assumes stoichiometric amounts of pterin and flavin. The extinction coefficient of the pterin chromophore, determined on the basis of its (p-aminobenzoyl)polyglutamate content (epsilon 360 = 25.7 x 10(3) M-1 cm-1), is in good agreement with that expected for a 5,10-methenyltetrahydrofolate derivative. Also consistent with this structure, the pterin chromophore could be reversibly hydrolyzed to yield a 10-formyltetrahydrofolate derivative or reduced to yield a 5-methyltetrahydrofolate derivative. The isolated enzyme could be reconstituted with various folate derivatives to yield enzyme that contained equimolar amounts of pterin and flavin. Similar results were obtained in reconstitution studies with the natural pterin chromophore, with 5,10-methenyltetrahydrofolate, and with 10-formyltetrahydrofolate. The results show that the polyglutamate moiety, previously identified in the natural chromophore, is not critical for binding. Reconstitution with the natural pterin chromophore did not affect catalytic activity. The latter is consistent with our previous studies which show that, although the pterin chromophore acts as a sensitizer in native enzyme, it is not essential for dimer repair which can occur at the same rate under saturating light with flavin (1,5-dihydro-FAD) as the only chromophore.
...
PMID:Reconstitution of Escherichia coli DNA photolyase with various folate derivatives. 265 34

Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyl-tetrahydrofolate (5,10-CH+-H4folate). Both chromophores are fluorescent, and either can function as a sensitizer in catalysis. At 77 K separate fluorescence emission bands are observed for FADH2 (lambda max = 505 nm, shoulder at 540 nm) and 5,10-CH+-H4folate (lambda max = 465, 440 nm) whereas at 5 degrees C only a shoulder at 505 nm is attributable to FADH2. Formation of an enzyme-substrate complex with various dimer-containing oligothymidylates [UV-oligo(dT)n] quenches the fluorescence due to FADH2 at 5 degrees C or 77 K and also stabilizes FADH2 against air oxidation. The fluorescence of 5,10-CH+-H4folate is unaffected by substrate. Reduction of the pterin chromophore eliminates the chromophore's fluorescence but does not affect catalytic activity or the ability of substrate to quench FADH2 fluorescence. Quenching of FADH2 fluorescence is fully reversible upon dimer repair. The results are consistent with the proposal that the singlet state of FADH2 functions as an intermediate in catalysis. Fluorometric titrations indicate that the enzyme has a similar affinity for dimers in UV-oligo(dT)4 (KD = 2.5 X 10(-7) M, delta G = 8.4 kcal/mol at 5 degrees C) or UV-oligo(dT)6, except for dimers located at the unphosphorylated 3' end of the oligomers where binding is considerably weaker.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Evidence for a singlet intermediate in catalysis by Escherichia coli DNA photolyase and evaluation of substrate binding determinants. 306 30

8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys293) in the flavin domain of Escherichia coli DNA photolyase to form an 8-(cysteinyl)FAD derivative covalently bound to the protein. About 80% protection against covalent attachment with 8-(methylsulfonyl)FAD was observed in the presence of an equimolar amount of FAD. Flavinylated photolyase retains the ability to repair pyrimidine dimers (15% of native activity) and to bind its antenna chromophore, 5,10-methenyltetrahydrofolate. Comparison of the properties of flavinylated enzyme with photolyase containing noncovalently bound 8-(methylthio)-FAD indicate that a perturbation is necessary to accommodate covalent bond formation. 8-(Methylthio)-FAD-reconstituted enzyme exhibits 95% of native activity. The aerobic stability of fully reduced and radical forms of 8-(methylthio)FAD enzyme is similar to that of native enzyme, whereas a radical form is not detected with flavinylated enzyme and the fully reduced enzyme is more easily oxidized by oxygen. The flavin in 8-(methylthio)FAD enzyme or flavinylated photolyase is shielded from solvent. However, the flavin environment in flavinylated enzyme is less hydrophobic as judged by spectral comparison with model 8-(alkylthio)flavins in various solvents. Enzyme containing noncovalently bound 8-(methylsulfonyl)-FAD was prepared by reconstitution with the fully reduced flavin which does not undergo covalent attachment. Covalent attachment was observed after reoxidation but probably involved dissociation and rebinding of oxidized 8-(methylsulfonyl)FAD. The results show that 8-(cysteinyl)FAD in flavinylated photolyase is at or near the normal flavin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Affinity probing of flavin binding sites. 2. Identification of a reactive cysteine in the flavin domain of Escherichia coli DNA photolyase. 791 92

A phr-gene from the filamentous fungus Neurospora crassa was overexpressed in Escherichia coli cells, yielding a biologically active photolyase. After purification till apparent homogeneity, the 66 kDa protein was found to contain equimolar amounts of 5,10-methenyltetrahydrofolic acid (MTHF) and FAD, classifying it as an MTHF-type photolyase. Compared to other MTHF photolyases the absorption maximum of Neurospora photolyase is shifted from ca 380 nm to 391 nm (epsilon = 34,800), while an additional shoulder is present at 465 nm. In dark-adapted enzyme the FAD chromophore is predominantly present in the oxidized form, in contrast with E. coli and Saccharomyces cerevisiae photolyase, which contain mainly semiquinone or fully reduced FAD, respectively. Preillumination or dithionite treatment converted oxidized FAD in Neurospora photolyase into the fully reduced form, with a concomitant shift of the absorption maximum from 391 to 396 nm and disappearance of the 465 nm shoulder. The action spectrum of photoreactivation coincides with the absorption spectrum of preilluminated (reduced) photolyase, extending the spectral region of MTHF-type photolyases from 380 till 396 nm. A quantum yield of 0.57 was obtained for the overall repair reaction. Comparison of spectral properties of FAD in Neurospora photolyase and the model compound lumiflavin points to an apolar microenvironment of photolyase-bound FAD. Neurospora photolyase has distinct advantages over E. coli photolyase as it is more stable and contains a full complement of chromophores.
...
PMID:DNA photolyase from the fungus Neurospora crassa. Purification, characterization and comparison with other photolyases. 793 8

The gene for the apoenzyme of Bacillus firmus photolyase was cloned and sequenced. The enzyme was overproduced in Escherichia coli, purified, and characterized. It has the unique property of having the maximum activity over a wavelength range where all other known photolyases exhibit modest activity. The enzyme contains reduced FAD and methenyltetrahydrofolate and has an absorption and action spectrum peak at 410 nm, and it repairs DNA with a quantum yield of phi approximately 0.75.
...
PMID:Characterization of a medium wavelength type DNA photolyase: purification and properties of photolyase from Bacillus firmus. 803 61

Cryptochromes are flavoproteins which serve as blue-light receptors in plants, animals, fungi and prokaryotes and belong to the same protein family as the catalytically active DNA photolyases. Cryptochrome 3 from the plant Arabidopsis thaliana (cry3; 525 amino acids, 60.7 kDa) is a representative of the novel cryDASH subfamily of UV-A/blue-light receptors and has been expressed as a mature FAD-containing protein in Escherichia coli without the signal sequence that directs the protein into plant organelles. The purified cryptochrome was found to be complexed to methenyltetrahydrofolate as an antenna pigment. Crystals of the cryptochrome-antenna pigment complex were obtained by vapour diffusion and display orthorhombic symmetry, with unit-cell parameters a = 76.298, b = 116.782, c = 135.024 A. X-ray diffraction data were collected to 1.9 A resolution using synchrotron radiation. The asymmetric unit comprises a cry3 dimer, the physiological role of which remains to be elucidated.
...
PMID:Crystallization and preliminary X-ray analysis of cryptochrome 3 from Arabidopsis thaliana. 1651 Dec

UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.
...
PMID:Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor. 1710 88

Cryptochromes are almost ubiquitous blue-light receptors and act in several species as central components of the circadian clock. Despite being evolutionary and structurally related with DNA photolyases, a class of light-driven DNA-repair enzymes, and having similar cofactor compositions, cryptochromes lack DNA-repair activity. Cryptochrome 3 from the plant Arabidopsis thaliana belongs to the DASH-type subfamily. Its crystal structure determined at 1.9 Angstroms resolution shows cryptochrome 3 in a dimeric state with the antenna cofactor 5,10-methenyltetrahydrofolate (MTHF) bound in a distance of 15.2 Angstroms to the U-shaped FAD chromophore. Spectroscopic studies on a mutant where a residue crucial for MTHF-binding, E149, was replaced by site-directed mutagenesis demonstrate that MTHF acts in cryptochrome 3 as a functional antenna for the photoreduction of FAD.
...
PMID:Cryptochrome 3 from Arabidopsis thaliana: structural and functional analysis of its complex with a folate light antenna. 1718 99


1 2 Next >>

---